The Alphavirus genus in the Togaviridae family includes a number of important human and animal pathogens, including Venezuelan (VEEV), eastern (EEEV) and western equine encephalitis (WEEV) viruses. The latter constitute a serious public health threat in the US, because they continuously circulate in nature and have the ability to cause fatal disease in humans and horses. In addition, EEEV, WEEV and, in particular, VEEV have the potential for use by terrorists and as biological warfare agents. In spite of the continuous threat of VEEV epidemics, the biology of VEEV, in particular the molecular basis of its high virulence, has been studied less intensively than that of other alphaviruses. It was believed that Sindbis (SINV) and Semliki Forest (SFV) viruses, which are dramatically less pathogenic than VEEV, represent good models for studying the mechanism of VEEV replication and virus-host interactions. However, results from recent comparative studies with VEEV and SINV suggested that VEEV replication and its pathogenesis strongly differ from those previously described for SINV. Hence, a great part of our knowledge about SINV biology cannot be directly applied to VEEV. Our preliminary studies strongly suggested that the VEEV capsid plays critical roles in inhibiting both nucleocytoplasmic trafficking and cellular transcription and, thus, downregulation of the innate immune response. Our results also suggested the 5'UTR of the VEEV genome is one of the important determinants of viral pathogenesis. Therefore, the proposed research is aimed at further understanding the functions of VEEV capsid and 5'UTR in virus replication, and in determining virus pathogenesis at different levels. The proposed experiments are organized into three aims. In specific aim 1, we intend to define functioning of VEEV capsid in the nucleocytoplasmic transport inhibition. In specific aim 2, we will perform a detailed investigation of the effects of the strain-specific mutations in the 5'UTR sequence on VEEV replication and pathogenesis on molecular and cellular levels; and in specific aim 3, we will apply already available information and new data that will be generated in our proposed experiments to develop new, highly attenuated strains of VEEV for vaccine applications. The specific aims represent three independent lines of research, but they are expected to generate complementary data. The results of this study will also be applicable to other encephalitogenic alphaviruses, such as EEEV and WEEV. PUBLIC HEALTH RELEVANCE: Alphaviruses comprise a group of widely distributed human and animal pathogens; some of them induce highly debilitating diseases and represent a serious public health threat in the US. The goal of this application is to understand the role of viral proteins in modification of the intracellular environment during virus replication and development of new, highly attenuated, live vaccines against encephalitogenic alphaviruses.